Abstract
It has been documented that the genotypic traits in symptomatic and asymptomatic cases of visceral leishmaniasis (VL) may be different. The current study aimed to find out and compare the genotype and intraspecies diversity of Leishmania Internal Transcribed Spacer 1 (ITS1) from asymptomatic and symptomatic VL cases in southern Iran. Methods. Buffy coat samples from seven VL patients, with clinical signs and symptoms, and seven asymptomatic VL cases, were evaluated in this study. Samples of asymptomatic individuals were obtained from children living in a VL endemic area in southern Iran, while the samples of symptomatic subjects were obtained from patients admitted to hospitals with a diagnosis of VL. DNA was extracted from the buffy coats of the samples and PCR-amplified, targeting the ITS1of Leishmania. The PCR products were sequenced, and the consensus sequences were assembled and multiple-aligned with a set of Leishmania strains retrieved from the GenBank, using Clustal W. The phylogenetic tree was rooted, using MEGAX software, and the diversities based on haplotype and nucleotides, as well as the number of polymorphic sites, were measured using DnaSP v5.0 software. The results of ITS1 sequencing in 5 out of 7 asymptomatic VL cases showed 99.25% to 100% similarity with the Leishmania infantum ITS1 sequence (accessed number: MN648746), and one isolate was considered as just Leishmania sp. In one sample, 99.75% similarity was seen with the ITS1 sequence of Crithidia fasciculata. Of the symptomatic VL patients, the PCR product revealed a 340 bp band corresponding to L. infantum in all of the samples. By analyzing the ITS1 sequences, all seven sequences formed a clade somewhat different from other Leishmania species and considered as Leishmania sp. Haplotype and nucleotide diversity were much more prevalent in symptomatic cases where six haplotypes were seen in the ITS1 of Leishmania from symptomatic patients and only two haplotypes were observed in the samples from asymptomatic cases. The findings of the current study showed that the Leishmania ITS1 from symptomatic VL and asymptomatic cases has significant genetic differences. Besides, infection with Crithidia fasciculata was reported, for the first time, in an asymptomatic case, which deserves further study.
Highlights
Leishmaniasis continues to be one of the health challenges in many countries of the world, with an estimated annual worldwide incidence of 700,000 to 1,300,000 for cutaneous leishmaniasis (CL) and approximately 500,000 cases for visceral leishmaniasis (VL) [1, 2]. e disease is caused by different species of Leishmania parasites and can produce a range of clinical symptoms based on the type of parasite involved [3, 4]. e spectrum begins in a general category of localized or diffuse skin involvement, which is a self-limiting skin condition
Most of the seroconverted asymptomatic VL cases do not develop an acute form of the disease, it has been shown that the progression to the overt form of the disease is associated with high positive qPCR, as well as rK39 ELISA or DAT antibody titers [17]
Informed consent was obtained from the patients or their guardians in the case of children. e subjects of this study were two groups of people. e first group consisted of 7 children admitted to Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran, with clinical signs and symptoms of the disease. ese VL patients had more than two weeks of fever, had hepatosplenomegaly, and they were all positive in IFAT, using an Iranian strain of L. infantum (MCAN/IR/14/M14) as an antigen and confirmed by a real-time PCR and rK39-rapid
Summary
Leishmaniasis continues to be one of the health challenges in many countries of the world, with an estimated annual worldwide incidence of 700,000 to 1,300,000 for cutaneous leishmaniasis (CL) and approximately 500,000 cases for visceral leishmaniasis (VL) [1, 2]. e disease is caused by different species of Leishmania parasites and can produce a range of clinical symptoms based on the type of parasite involved [3, 4]. e spectrum begins in a general category of localized or diffuse skin involvement, which is a self-limiting skin condition. In VL, most cases do not progress to overt disease and remain asymptomatic [5,6,7]. Most of the seroconverted asymptomatic VL cases do not develop an acute form of the disease, it has been shown that the progression to the overt form of the disease is associated with high positive qPCR, as well as rK39 ELISA or DAT antibody titers [17]. It has been suggested that healthy people living in given VL endemic areas having high antibody titers or being qPCR positive have an increased chance to progress to VL disease [17]. QPCR on blood samples showed 500 times less parasitemia in asymptomatic cases in comparison with the symptomatic ones in India [12] It has been suggested that healthy people living in given VL endemic areas having high antibody titers or being qPCR positive have an increased chance to progress to VL disease [17]. qPCR on blood samples showed 500 times less parasitemia in asymptomatic cases in comparison with the symptomatic ones in India [12]
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